Structural Deterioration in Produce: Phospholipase D, Membrane Deterioration, and Senescence - Postharvest Biology and Technology of Fruits, Vegetables, and Flowers - page 211

strawberry fruit membranes may also be due to PLD alpha. Western blot analysis of straw-
berry membrane proteins using polyclonal antibodies raised against rapeseed PLD alpha
(Novotna et al., 1999) showed a predominant cross reactivity with a 92-kDa protein (Yuan
et al., 2005), and nothing above this molecular mass, suggesting that PLD beta (112 kDa)
and gamma (98 kDa) isoforms may occur below detectable levels, or do not react with
the PLD alpha antibodies. As well, the PLD alpha 1 encoded by FaPLD α 1 from straw-

berry fruit (Yuan et al., 2005) is very similar to that encoded by LePLD 1 from tomato.

In general, strawberry PLD alpha is also similar to tomato PLD alpha in its properties and
regulation by calcium (Tiwari et al., unpublished). Despite differences in kinetic properties,
strawberry fruit mitochondrial PLD appears to be similar to microsomal PLD in its molec-
ular mass, about 92 kDa, which is indicative of an alpha-type PLD (Yuan et al., 2006a).
The different kinetic properties of mitochondrial versus microsomal PLD may arise from
posttranslational modifications, or the presence/absence of particular modulatory lipids or
other effectors. Irrespective of whether a fruit is climacteric (tomato), weakly climacteric
(hybrid honeydew melon), or nonclimacteric (strawberry), PLD may play a key role in the
initiation and progression of ripening and senescence.
Three isogenes encoding PLD alphas (referred to here as LePLD

α

α 1 , LePLD α 2 , and

LePLD 1

(Almquist and Paliyath, 2000; GenBank accession number AF201661) was cloned us-
ing a novel strategy for the design of primers known as the CODEHOP program (Rose
et al., 1998), by which PCR primers were designed based on highly conserved regions of
Arabidopsisthaliana PLD alpha-, beta-, and gamma-encoded amino acid sequences aligned
using the Clustal W program (Altschul et al., 1997). A CODEHOP primer is degenerated at
the 3 core region, with a length of 11-12 bp spanning four codons representing highly con-
served amino acids, and nondegenerate at the 5 consensus clamp region, which is variable
in length. The hybrid structure (5 consensus and 3 degenerate) of CODEHOP primers has
been reported to result in specific amplification of cDNA during early cycles of PCR, and
selective amplification of products during later cycles

α 3 ) have been cloned from tomato and further studied (Table 9.2). LePLD α

Forward and reverse primers were
generated and PCR performed using tomato root cDNA (reported in Pinhero et al., 2003).
Initially this step amplified two cDNA fragments: one 883-bp long corresponding to a PLD
alpha and another 936-bp long corresponding to a PLD beta or second PLD alpha. Forward

.

Table 9.2 Percent deduced amino acid sequence identity/similarity among seven PLD alpha full-length
cDNAs from tomato ( LePLD

α 1 , LePLD α 2 , and LePLD α 3 ), strawberry ( FaPLD α 1 ), honeydew melon

( CmPLD α 1 and CmPLD α 2 ), and cucumber ( CsPLD α 1)

LePLD α 1 LePLD α 2 LePLD α 3 FaPLD α 1 CmPLD α 1 CmPLD α 2 CsPLD α 1

LePLD α 1 — 76/87 74/86 81/89 82/92 75/98 81/91

LePLD α 2 76/87 — 87/94 75/87 76/87 74/87 76/87

LePLD α 3 74/86 87/94 — 73/86 74/86 71/86 74/86

FaPLD α 1 81/89 75/87 73/86 — 82/91 76/88 82/91

CmPLD α 1 82/92 76/87 74/86 82/91 — 78/90 97/98

CmPLD α 2 75/88 74/87 71/86 76/88 78/90 — 77/89

CsPLD α 1 81/91 76/87 74/86 82/91 97/98 77/89 —

The corresponding GenBank protein accession numbers are as follows: LePLD α 1 = AAF17557, LePLD α 2

= AAG48162, LePLD α 3 = AAG45486, FaPLD α 1 = AAW83125, CmPLD α 1 = ABB82551, CmPLD α 2 =

ABS86615, CsPLD α 1 = ABN13537.